ES2628135T3 - Human antibodies for protein 4 similar to human angiopoietin - Google Patents

Abstract

An isolated human antibody or antigen-binding fragment thereof that specifically binds to human angiopoietin-like protein 4, comprising: (a) a heavy chain complexity determining region 1 / heavy chain complementarity determining region 2 / heavy chain complementarity determining region 3 having the combination of amino acid sequences of SEQ ID NO: 28/30/32; and (b) a light chain complementarity determining region 1 / light chain complementarity determining region 2 / light chain complementarity determining region 3 having the combination of amino acid sequences of SEQ ID NO: 36/38/40 .

Description

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DESCRIPTION

Human antibodies to protein 4 similar to human angiopoietin Field of the invention

The present invention relates to human antibodies and fragments that bind to human antibody antigens that specifically bind to human angiopoietin-like protein 4 (hANGPTL4) and to antibodies for use in therapeutic methods.

State of the art related statement

Lipoprotelna lipase (LPL) has a central role in the metabolism of lipoprotelnas to maintain normal levels of lipoprotelnas in the blood and, through specific regulation in the tissue of its activity, to determine when and in which tissues triglycerides are discharged (TG). It has been reported that ANGPTL4 inhibits LPL and retards the catabolism of lipoprotelnas, in humans and rodents. Mice without ANGPTL4 exhibit a significant decrease in serum TG. In contrast, an injection of ANGPTL4 in mice produces a rapid increase in circulating lipids and this is at a higher rate than the injection of prothena 3 similar to angiopoietin (ANGPTL3) (Yoshida et al., 2002, J Lipid Res 43 : 1770-1772). It is known that the spiral coil region of the N-terminal, not the fibrinogen-like domain of the C-terminal, of ANGPTL4, is important in the inhibition of LPL activity and, therefore, for the indication of hypertriglyceridemia. These observations indicate that the inhibition of ANGPTL4 could be beneficial in the treatment of diseases characterized by elevated levels of lipids, including primary dyslipidemia and hypertriglyceridemia associated with obesity, metabolic syndrome, type II diabetes and the like. ANGPTL4 has also been implicated for having a role in angiogenesis and cancer (Galaup et al., 2006, PNAS 103 (49): 18721-18726, Kim et al., 2000, Biochem J 346: 603-610, and Ito y collaborators, 2003, Cancer Res 63 (20): 6651-6657).

Nucleic acid and amino acid sequences of human ANGPTL4 are shown in SEQ ID NOS: 475 and 476, respectively. Antibodies to ANGPTL4 are disclosed, for example, in WO 2006/074228 and WO 2007/109307.

Brief Summary of the Invention

The invention provides fully human monoclonal antibodies (mAbs) and antigen-binding fragments thereof that specifically bind and neutralize the activity of human ANGPTL4 (hANGPTL4).

Antibodies (Ab) may be full length (for example, an IgG1 or IgG4 antibody) or may comprise only a portion of antigen binding (for example, a Fab, F (ab ') 2 or scFv fragment), and may be modified to affect functionality, for example, to eliminate residual effector functions (Reddy et al., 2000, J. Immunol., 164: 1925-1933).

Specifically, the present invention provides an isolated human antibody or antigen binding fragment thereof that specifically binds to human angiopoietin-like protein 4 (hANGPTL4), comprising:

(a) a heavy chain complementarity determining region 1 (HCDR1) / HCDR2 / HCDR3 having the amino acid sequence combination of SEQ ID NO: 28/30/32; Y

(b) a light chain complementarity determination region 1 (LCDR1) / LCDR2 / LCDR3 having the amino acid sequence combination of SEQ ID NO: 36/38/40.

The invention also provides an antibody or antigen binding fragment of an antibody comprising a heavy chain variable region (HCVR) selected from SEQ ID NO: 487, 26, 42 and 46.

The invention further provides an antibody or antigen binding fragment comprising a light chain variable region (LCVR) selected from SEQ ID NO: 44, 34 and 48.

The invention also provides an antibody or fragment thereof comprising a pair of HCVR and LCVR (HCVR / LCVR) sequences selected from SEQ ID NO: 487/44, 26/34, 42/44 and 46/48.

The invention also comprises an antibody or antigen-binding fragment of an antibody that specifically binds to hANGPTL4, wherein the antibody or fragment thereof comprises the heavy and light chain CDR domains contained within a HCVR / LCVR pair of SEQ ID No: 487/44, 26/34, 42/44 and 46/48.

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Methods and techniques for identifying the CDRs within the amino acid sequences of HCVR and LCVR are known in the art and can be applied to identify the CDRs within the specified amino acid sequences of HCVR and / or LCVR disclosed herein. Conventional definitions that can be applied to identify the limits of the CDRs include the definition of Kabat, the definition of Chothia and the definition of AbM. In general terms, the definition of Kabat is based on sequence variability, the definition of Chothia is based on the location of the structural regions of the loop and the definition of AbM is a compromise between the approaches of Kabat and Chothia. See, for example, Kabat, "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273: 927-948 (1997); and Martin et al., Proc. Natl Acad. Sci. USA 86: 9268-9272 (1989). Public databases are also available to identify the CDR sequences within an antibody.

The invention also provides an isolated nucleic acid molecule encoding anti-ANGPTL4 antibodies or fragments thereof as described above. Recombinant expression vectors carrying the nucleic acids of the invention, and host cells, for example, bacterial cells, such as E. coli or mammalian cells, such as CHO cells, into which such vectors have been introduced, also They are encompassed by the invention as well! as the methods to produce the antibodies by culturing the host cells under conditions that allow the production of the antibodies and recovering the antibodies produced.

Antibodies or antigen-binding fragments thereof are provided that specifically bind hANGPTL4 with an equilibrium dissociation constant (Kd) of approximately 1 nM or less, measured by the surface plasmon resonance assay (e.g. BIACOREMR) . The antibodies may exhibit a Kd of approximately 500 pM or less; approximately 400 pM or less; approximately 300 pM or less; approximately 200 pM or less; approximately 150 pM or less; approximately 100 pM or less; or about 50 pM or less.

An anti-hANGPTL4 antibody or antigen-binding fragment thereof is provided that binds to the hANGPTL4 protein of SEQ ID NO: 476, but does not cross-react with a related protein, such as a human-like angiopoietin-like protein (hANGPTL3; SEQ ID NO: 485), as determined, for example, by ELISA, surface plasmon resonance assay, or LUMINEX® XMAP® Technology, as described herein. ANGPTL3 is another secreted protein known to reduce the activity of the LPL and has a spiral coil region at the N-terminus and a fibrinogen-like domain at the C-terminus (Ono et al., 2003, J Biol Chem 43: 41804-41809). An anti-hANGPTL4 antibody or antigen binding fragment thereof can bind to a hANGPTL4 protein and cross-react with a hANGPTL3 protein. The binding affinity of the hANGPTL4 antibody or fragment thereof to the hANGPTL3 protein may be about 75% or less, or about 50% or less, of the binding affinity of the antibody or fragment to the hANGPTL4 protein. The anti-hANGPTL4 antibody or its antigen binding fragment may not cross-react with mouse ANGPTL4 (mANGPTL4: SEQ ID NO: 478), but may cross-react with monkey cynomolgus ANGPTL4 (Macaca fascicularis, the sequence of amino acids from residues 1-148 of the N-terminus and the coding DNA sequences are shown as SEQ ID NOS: 490 and 489, respectively) and / or from Rhesus monkeys (Macaca mulatta), the amino acid sequence of residues 1 -148 N-terminal end and DNA coding sequences are shown as SEQ ID NOS: 492 and 491, respectively).

Anti-hANGPTL4 antibodies that have a modified glycosylation pattern are also contemplated. In some applications, modification to eliminate undesirable glycosylation sites may be useful, or, for example, the elimination of a fucose fraction to increase the function of antibody-dependent cellular cytotoxicity (ADCC) (see Shield et al. (2002) JBC 277: 26733). In other applications, removal of the N-glycosylation site may reduce undesirable immune reactions against therapeutic antibodies or increase antibody affinities. In still other applications, galactosylation modification can be performed to modify complement dependent cytotoxicity (CDC).

The invention also presents a pharmaceutical composition comprising a recombinant human antibody or fragment thereof of the invention that specifically binds to hANGPTL4 and a pharmaceutically acceptable carrier. The invention also presents a composition that is a combination of an antibody or fragment thereof binding to the antigen of the invention, and a second therapeutic agent. The second therapeutic agent may be one or more of any agent such as (1) 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, such as cerivastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, fluvastatin , lovastatin, pravastatin and the like; (2) cholesterol captation inhibitors and / or bile acid reabsorption; (3) niacin, which increases the catabolism of lipoprotelnas; (4) amphipathic carboxylic fibrates or acids that reduce the level of low density lipoprotelns (LDL), improve levels of high density lipoprotelns (HDL) and TG and reduce the number of non-fatal cardiac attacks; and (5) activators of the transcription factor LXR that plays a role in cholesterol elimination, such as 22-hydroxycholesterol or fixed combinations such as ezetimibe plus simvastatin; a statin with a biliary resin (eg, cholestyramine, colestipol, colesevelam), a fixed combination of niacin plus a statin (for example, niacin with lovastatin); or with other lipid lowering agents such as omega 3 fatty acid esters (eg, omacor). In addition, the

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Second therapeutic agent may be one or more ANGPTL4 inhibitors, as! as inhibitors of other molecules, such as ANGPTL3, ANGPTL5, ANGPTL6 and proprotelna convertase subtilisin / kexin type 9 (PCSK9), which are involved in lipid metabolism, in particular, homeostasis of cholesterol and / or triglycerides. Inhibitors of these molecules include small molecules and antibodies that specifically bind to these molecules and block their activity.

The second therapeutic agent may be one or more anticancer agents, such as chemotherapeutic agents, antiangiogenic agents, growth inhibiting agents, cytotoxic agents, apoptotic agents and other agents well known in the art for treating cancer or other proliferative diseases or disorders, as well. ! as other therapeutic agents, such as analgesics, anti-inflammatory agents, including non-steroidal anti-inflammatory drugs (NSAIDS), such as Cox-2 inhibitors, in order to improve and / or reduce the symptoms that accompany the underlying cancer / tumor.

The invention also features the anti-hANGPTL4 antibody or fragment of the invention for use in a method for improving, alleviating, inhibiting or preventing a disease or disorder mediated by ANGPTL4, wherein the disease or disorder mediated by ANGPTL4 is selected from hypertriglyceridemia, hypercholesterolemia. , chylomicronemia, atherogenic dyslipidemia, cardiovascular disease or disorder, acute pancreatitis, non-alcoholic steatohepatitis, (NASH), diabetes and obesity. Examples of diseases or disorders that can be treated using the antibodies disclosed herein include, but are not limited to, those that involve lipid metabolism, such as hyperlipidemia, hyperlipoproteinemia and dyslipidemia, including atherogenic dyslipidemia, diabetic dyslipidemia, hypertriglyceridemia, including hypertriglyceridemia. severe with TG> 1,000 mg / dL, hypercholesterolemia, chylomicronemia, mixed dyslipidemia (obesity, metabolic syndrome, diabetes, etc.), lipodystrophy, lipoatrophy and the like, which are caused, for example, by a decrease in the activity of LPL and / or LPL deficiency, lower LDL receptor activity and / or LDL receptor deficiency, altered ApoC2, ApoE deficiency, ApoB increase, production increase and / or decrease in the elimination of very low density lipoprotin (VLDL) ), certain drug treatments (for example, dyslipidemia induced by glucocorticoid treatment), any genetic predisposition, diet, lifestyle. Antibodies can also prevent or treat diseases or disorders associated with or resulting from hyperlipidemia, hyperlipoproteinemia and / or dyslipidemia, including, but not limited to, cardiovascular diseases or disorders, such as atherosclerosis, aneurysm, hypertension, angina, stroke, cerebrovascular diseases, congestive heart failure, coronary heart disease, myocardial infarction, peripheral vascular diseases; acute pancreatitis; non-alcoholic steatohepatitis (NASH); blood sugar disorders, such as diabetes; obesity.

Other examples of diseases or disorders that can be treated by antibodies include cancer / tumors as! such as non-neoplastic diseases or disorders associated with angiogenesis, including ocular angiogenic diseases or disorders, such as age-related macular degeneration, central retinal vein occlusion or branched retinal vein occlusion, diabetic retinopathy, prematurity retinopathy, diseases or inflammatory disorders, such as arthritis, rheumatoid arthritis (RA), psoriasis.

The invention also provides the use of an antibody or fragment thereof binding to the antigen of the invention in the manufacture of a medicament for use to improve, relieve, inhibit or prevent a disease or disorder mediated by ANGPTL4, wherein the disease or disorder ANGPTL4-mediated is selected from hypertriglyceridemia, hypercholesterolemia, chylomicronemia, atherogenic dyslipidemia, cardiovascular disease or disorder, acute pancreatitis, non-alcoholic steatohepatitis, diabetes and obesity.

Other embodiments will be apparent from a review of the following detailed description.

Brief description of the figures

Figure 1 shows an alignment of heavy chain variable region (HCVR) sequences of the H1H268P, H1H284P, H1H291P and H1H292P antibodies.

Figure 2 shows an alignment of light chain variable region (LCVR) sequences of the H1 H268P, H1 H284P, H1 H291P and H1 H292P antibodies.

Figures 3A and 3B show the pharmacokinetic elimination of anti-ANGPTL4 antibodies in wild-type mice (Figure 3A) and in transgenic mice expressing human ANGPTL4 [hAngptl4 (+/ +) mice; or "humanized ANGPTL4 mice"] (Figure 3B). H4H268P2 (□); H4H284P (▲); and the control of hlgG4 (•).

Figure 4 shows the effect of the anti-ANGPTL4 antibody, H4H268P2, on serum triglyceride (TG) levels in humanized ANGPTL4 mice crossed with null mice in ApoE. The percentage changes (%) of serum TG levels by H4H268P2 are shown, compared to the control antibody with irrelevant specificity. Ab of control (o); and H4H268P2 (■).

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Figure 5 shows the effects of the anti-ANGPTL4 H4H268P2 antibody and the TG fenofibrate reducing drug, each alone or in combination, on serum TG levels in humanized ANGPTL4 mice.

Figure 6 shows the results of the phase I pilot study on the effects of anti-ANGPTL4 antibodies on fasting serum TG levels, among others, on obese Rhesus monkeys (Macaca mulatta). All monkeys received an intravenous (IV) infusion of vehicle (10 mM histidine, pH 6) on day 5 and H4H268P2 (n = 3) (•) or H4H284P (n = 3) (□) at the rate of 10 mg / kg IV on Day 0. Serum samples were collected from baseline to Day 35 after dosing. The average baseline for each animal was determined based on the samples taken in days -7, -5 and 0, and the percentage changes (%) of serum TG levels of the baseline determined and averaged for each group of Ab.

Figure 7 shows the results of the phase II pilot study on the effects of anti-ANGPTL4 H4H268P2 antibody on fasting serum TG levels in obese monkeys, as described for Figure 6, except that the infusion stage was omitted of vehicle. The reference mean was obtained for each monkey based on the samples taken in days -7, -3 and 0. The monkeys were divided into groups based on their baselines: A. TG <150 mg / dL (n = 3; □); B. 150 mg / dL <TG <500 mg / dL (n = 4; •); and C. TG> 1,000 mg / dL (n = 1; V). The percentage changes (%) of the fasting TG levels of the baseline for each monkey were determined and averaged for each group.

Error bars in all graphs indicate the mean ± SEM.

Detailed Description Definitions

The term "protelna 4 similar to human angiopoietin" or "hANGPTL4", as used herein, refers to hANGPTL4 having the nucleic acid sequence shown in SEQ ID NO: 475 and the amino acid sequence of the SEQ ID NO: 476, or a biologically active fragment thereof.

The term "antibody", as used herein, refers to immunoglobulin molecules composed of four polypeptide chains, two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (HCVR) and a heavy chain constant region (Ch, which comprises domains Ch1, Ch2 and Ch3). Each light chain is composed of a variable light chain region (LCVR) and a constant light chain region (CL). The HCVR and LCVR can be subdivided into regions of hypervariability, called complementarity determining regions (CDR), interspersed with more conserved regions, called structural regions (FR). Each HCVR and LCVR consists of three CDRs and four FRs, arranged from the amino terminal end to the carboxy terminal end in the following order: fR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

It is also possible to substitute one or more CDR residues or omit one or more of the CDRs. Antibodies have been described in the scientific literature in which one or two CDRs can be dispensed with for binding. Padlan et al. (1995, FASEB J. 9: 133-139) analyzed the contact regions between the antibodies and their antigens, based on the published crystalline structures, and concluded that only about one-fifth to one-third of the CDR residues actually They make contact with the antigen. Padlan also found many antibodies in which one or two CDRs did not have amino acids in contact with an antigen (see also Vajdos et al., 2002, J Mol Biol 320: 415-428).

CDR residues that are not in contact with the antigen can be identified based on previous studies (for example, H60-H65 residues in CDRH2 are not often required), from Kabat CDR regions located outside the Chothia CDR , by molecular modeling and / or empirically. If a CDR or residue or residues thereof is omitted, it is usually substituted with an amino acid that occupies the corresponding position in another human antibody sequence or a consensus of said sequences. Positions for substitution within CDRs and amino acids for substitution can also be selected empirically. Empirical substitutions may be conservative or non-conservative substitutions.

The term "human antibody", as used herein, is intended to include antibodies that have variable and constant regions derived from human germline immunoglobulin sequences. The human mAbs of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (for example, mutations introduced by random or specific site mutagenesis in vitro or by somatic mutation in vivo), for example, in the CDRs and in particular CDR3. However, the term "human antibody", as used herein, is not intended to include mAbs into which CDR sequences derived from the germ line of other mammalian species (eg, mouse) have been grafted, in human FR sequences.

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The fully human anti-hANGPTL4 antibodies described herein may comprise one or more amino acid substitutions, insertions and / or deletions in the structural and / or CDR regions of the heavy and light chain variable domains compared to the corresponding sequences of germ line. Such mutations can be readily determined by comparing the amino acid sequences disclosed herein with available germline sequences, for example, from public databases of antibody sequences. The present invention includes antibodies and antigen binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, in which one or more amino acids within one or more structural regions and / or CDR are mutated to the corresponding residue or residues of the germline sequence from which the antibody was derived, or to the corresponding residue or residues of another human germline sequence or to a conservative amino acid substitution of the corresponding germline residue or residues (said Sequence changes are collectively referred to as "germline mutations"). A person skilled in the art, beginning with the sequences of variable regions of heavy and light chain disclosed herein, can easily produce numerous antibodies and antigen-binding fragments comprising one or more individual reverse germline mutations or combinations. from the same. All residues of the structure and / or CDR within the Vh and / or Vl domains can be mutated again to the residues found in the original germ sequence from which the antibody was derived. Alternatively, only certain residues are mutated back to the original germline sequence, for example, only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within of CDR1, CDR2 or CDR3. One or more of the structure and / or CDR residues may be mutated to the corresponding residue or residues of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived). In addition, the antibodies of the present invention may contain any combination of two or more germline mutations within the structural and / or CDR regions, for example, where certain individual residues are mutated to the corresponding residues of a germline sequence. particular while certain other residues other than the original germline sequence are maintained or mutated to the corresponding residue of a different germline sequence. Once obtained, antibodies and antigen-binding fragments that contain one or more germline mutations can easily be tested for one or more desired properties, such as, improved binding specificity, increased binding affinity, antagonistic biological properties or enhanced or enhanced agonists (as the case may be), reduced immunogenicity, etc. Antibodies and antigen binding fragments obtained in this general manner are encompassed within the present invention.

The present description also provides anti-ANGPTL4 antibodies comprising variants of any of the amino acid sequences of HCVR, LCVR and / or CDR disclosed herein that have one or more conservative substitutions. For example, the present disclosure includes anti-ANGPTL4 antibodies having amino acid sequences of HCVr, LCVR and / or CDR, for example, with 10 or less, 8 or less, 6 or less, 4 or less, 2 or 1, substitution or conservative amino acid substitutions with respect to any of the amino acid sequences of HCVR, LCVR and / or CDR disclosed herein. An HCVR may comprise the amino acid sequence of SEQ ID NO: 487 with 10 or less conservative amino acid substitutions therein. An HCVR may comprise the amino acid sequence of SEQ ID NO: 487 with 8 or less amino acid conservative substitutions therein. An HCVR may comprise the amino acid sequence of SEQ ID NO: 487 with 6 or less conservative amino acid substitutions therein. An HCVR may comprise the amino acid sequence of SEQ ID NO: 487 with 4 or less conservative amino acid substitutions therein. An HCVR may also comprise the amino acid sequence of SEQ ID NO: 487 with 2 or 1 substitution or conservative amino acid substitutions therein. An LCVR may comprise the amino acid sequence of SEQ ID NO: 44 with 10 or less conservative amino acid substitutions therein. An LCVR may comprise the amino acid sequence of SEQ ID NO: 44 with 8 or less amino acid conservative substitutions therein. An LCVR may comprise the amino acid sequence of SEQ ID NO: 44 with 6 or less amino acid conservative substitutions therein. An LCVR may comprise the amino acid sequence of SEQ ID NO: 44 with 4 or less conservative amino acid substitutions therein. An LCVR may comprise the amino acid sequence of SEQ ID NO: 44 with 2 or 1 substitution or conservative amino acid substitutions therein.

Unless specifically indicated otherwise, it will be understood that the term "antibody", as used herein, encompasses antibody molecules comprising two heavy chains of immunoglobulin and two light chains of immunoglobulin (ie, "molecules of complete antibody "), as! as fragments thereof binding to the antigen. The terms "antigen binding portion" of an antibody, "antigen binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically modified polypeptide or glycoproteln of natural origin. which specifically binds to an antigen to form a complex. Antigen binding fragments of an antibody can be derived, for example, from complete antibody molecules using any suitable standard technique such as proteolytic digestion or recombinant genetic engineering techniques that involve manipulation and expression of DNA encoding the variable domains and ( optionally) antibody constant. Said DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example,

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phage expression antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or through the use of molecular biology techniques, for example, to arrange one or more variable and / or constant domains in a suitable configuration, or introduce codons, create cystel residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen binding fragments include: (i) Fab fragments; (ii) F (ab ') 2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv molecules (scFv); (vi) dAb fragments; and (vii) minimum recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (eg, an isolated region determining complementarity (CDR)). Other modified molecules, such as diabodies, triabodies, tetrabodies and minibodies, are also encompassed within the expression "antigen binding fragment", as used herein.

An antigen-binding fragment of an antibody typically comprises at least one variable domain. The variable domain can be of any amino acid size or composition and will generally comprise at least one CDR that is adjacent to, or is in the frame with one or more structural sequences. In antigen-binding fragments that have a Vh domain associated with a Vl domain, the Vh and Vl domains may be located relative to each other in any suitable arrangement. For example, the variable region may be dimeric and contain the Vh-Vh, Vh-Vl or Vl-Vl dlmers. Alternatively, the antigen binding fragment of an antibody may contain a monomeric Vh or Vl domain.

An antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Examples of non-limiting configurations of variable and constant domains that can be found within an antigen-binding fragment of an antibody of the present invention include: (i) Vh-Ch1; (ii) Vh-Ch2; (iii) Vh-Ch3; (iv) Vh-Ch1-Ch2; (v) Vh-Ch1-Ch2-Ch3; (vi) Vh-Ch2-Ch3; (vii)

Vh-Cl; (viii) Vl-Ch1; (ix) Vl-Ch2; (x) Vl-Ch3; (xi) Vl-Ch1-Ch2; (xii) Vl-Ch1-Ch2-Ch3; (xiii) Vl-Ch2-Ch3; and (xiv) Vl-Cl. In

Any configuration of variable and constant domains, including any of the examples of configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. A hinge region may consist of at least 2 (for example, 5, 10, 15, 20, 40, 60 or more) amino acids that result in a flexible or semi-flexible bond between adjacent variable and / or constant domains in a single molecule of polypeptide. In addition, an antigen-binding fragment of an antibody of the present invention may comprise a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with each other and / or with one or more monomeric domains Vh or Vl (for example, via link or disulfide bonds).

As with complete antibody molecules, the antigen binding fragments can be monospecific or multispecific (eg, bispecific). A multispecific antigen-binding fragment of an antibody typically comprises at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or a different epltope on the same antigen. Any multispecific antibody format, including the examples of bispecific antibody formats described herein, can be adapted for use in the context of an antigen binding fragment of an antibody of the present invention using routine techniques available in the art. .

The antibody or antibody fragments of the invention may be conjugated to a therapeutic fraction ("immunoconjugate"), such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant or a radioisotope.

The term "specifically binds", or the like, means that an antibody or antigen binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. The specific union can be characterized by an equilibrium dissociation constant (Kd) of approximately 1x10‘6 M or less (that is, a smaller Kd indicates a firmer union). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance and the like. However, an isolated antibody that specifically binds to hANGPTL4 may exhibit cross-reactivity with other antigens, such as ANGPTL4 molecules of other species, for example, ANGPTL4 and / or cynomolgus monkey hANGPTL3 having the amino acid sequence of SEQ ID. NO: 485. In addition, multispecific antibodies (eg, bispecific) that bind to hANGPTL4 are considered and it is considered, however, that one or more additional antigens "specifically bind" to hANGPTL4, as used herein. .

The term "high affinity" antibody refers to those antibodies that have a binding affinity to hANGPTL4, expressed as Kd, of about 1 x 10'9 M or less, about 0.5 x 10'9 M or less, about 0.25 x 10'9 M or less, about 1 x 10_1 ° M or less, or about 0.5 x 10_1 ° M or less, as measured by surface plasmon resonance, for example, BIACOREMR or affinity ELISA in solution.

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The term "Kd", as used herein, refers to the equilibrium dissociation constant of a particular antibody-antigen interaction.

By the term "slowdown rate", "Kdisociation" or "kd" is meant an antibody that dissociates from hANGPTL4 with a rate constant of 1 x 10-3 s-1 or less, preferably 1 x 10-4 s- 1 or less, as determined by surface plasmon resonance, for example, BIACOREMR.

The term "intrinsic affinity constant" or "ka" means an antibody that is associated with hANGPTL4 with a velocity constant of approximately 1 x 103 M-1 s-1 or higher, as determined by surface plasmon resonance , for example, BIACOREMR.

An "isolated antibody", as used herein, refers to an antibody that is substantially free of other mAbs that have different antigenic specificities (for example, an isolated antibody that specifically binds to hANGPTL4 is substantially free of mAb that specifically bind antigens other than hANGPTL4). An isolated antibody that specifically binds to hANGPTL4 may, however, have cross-reactivity with other antigens, such as ANGPTL4 molecules of other species, such as cynomolgus monkey, and / or other related proteins, such as human ANGPTL3.

A "neutralizing antibody," as used herein (or an "antibody that neutralizes the activity of ANGPTL4"), refers to an antibody whose binding to ANGPTL4 results in the inhibition of at least one biological activity of ANGPTL4. . This inhibition of the biological activity of ANGPTL4 can be evaluated by measuring one or more indicators of biological activity of ANGPTL4 by one or more of several standard in vitro or in vivo assays known in the art (see also the examples below).

The term "surface plasmon resonance", as used herein, refers to an optical phenomenon that allows the analysis of biospecific interactions in real time by detecting alterations in the concentrations of proteins within a biosensor matrix, for example, using the BIACOREMR system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

The term "epltopo" is a region of an antigen that is bound by an antibody. Epltopos can be defined as structural or functional. Functional epltopos are generally a subset of structural epltopos and have those residues that contribute directly to the affinity of the interaction. Epltopos can also be conformational, that is, non-linear amino acid compounds. Epltopos may include determinants that are chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl groups or sulfonyl groups and, in certain circumstances, may have specific three-dimensional structural characteristics and / or specific loading characteristics.

The term "substantial identity" or "substantially identical" when referring to a nucleic acid or fragment thereof, indicates that, when it is optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary chain) , there is a nucleotide sequence identity at least about 90% and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, measured by any well-known sequence identity algorithm , such as FASTA, BLAST or GAP, as discussed below.

As it applies to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences, when optimally aligned, such as by GAP or BESTFlT programs that use weighting gaps by default, share at least one 90% sequence identity, even more preferably at least 95%, 98% or 99% sequence identity. Preferably, residue positions that are not identical are differentiated by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (group R) with similar chemical properties (eg, loading or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity can be adjusted upwards to correct the conservative nature of the substitution. The means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331. Examples of amino acid groups having side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) side chains containing amide: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) basic side chains: lysine, arginine and histidine; 6) acidic side chains: aspartate and glutamate, and 7) sulfur-containing side chains: cystel and methionine. Preferred amino acid conservative substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate and asparagine-glutamine. Alternatively, a conservative replacement is any change that has a positive value in the PAM250 log likelihood matrix described in Gonnet et al. (1992) Science

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256: 1443-45. A "moderately conservative" replacement is any change that has a non-negative value in the PAM250 logarithmic likelihood matrix.

Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as GAP and BESTFIT that can be used with predetermined parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides of different species of organisms or between a wild-type protein and a mutein of it. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA with predetermined or recommended parameters; a program in GCG Version 6.1. FASTA (for example, FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000), cited above). Another preferred algorithm when comparing a sequence of the invention with a database containing a large number of sequences from different organisms is the BLAST computer program, especially BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and (1997) Nucleic Acids Res. 25: 3389-402.

By the phrase "therapeutically effective amount" is meant an amount that produces the desired effect for which it is administered. The exact amount will depend on the purpose of the treatment, the age and size of a treated subject, the route of administration and the like, and will be verifiable by a person skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

Preparation of human antibodies

Methods for generating human antibodies in transgenic mice are known in the art. Any of these known methods can be used in the context of the present invention to make human antibodies that specifically bind ANGPTL4.

Using the VELOCIMMUNEMR technology or any other known method to generate monoclonal antibodies, high affinity chimeric antibodies for ANGPTL4 having a human variable region and a mouse constant region are initially isolated. As in the experimental section below, the antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, and the like.

In general, the antibodies of the present invention have very high affinities, which typically have a Kd of about 10-12 M to about 10-9 M, when measured by binding to the antigen, either immobilized in solid phase or in phase from solution. The mouse constant regions are replaced by desired human constant regions, for example, wild-type IgG1 (SEQ ID NO: 481) or IgG4 (SEQ ID NO: 482), or modified IgG1 or IgG4 (eg, sEq ID NO: 483), to generate the fully human antibodies of the invention. Although the constant region selected may vary according to the specific use, the binding characteristics of the high affinity antigen and target specificity of the antibodies reside in the variable region.

Epitope mapping and related technologies

For the detection of antibodies that bind to a particular epitope, a routine cross-block assay such as that described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY) can be performed. Other methods include alanine scan mutants, peptide transfers (Reineke (2004) Methods Mol Biol 248: 443-63), or analysis of peptide cleavage. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens (Tomer (2000) Protein Science 9: 487-496) can be employed.

The term "epitope" refers to a site on an antigen to which B and / or T cells respond. B cell epitopes can be formed from both contiguous amino acids and non-contiguous amino acids juxtaposed by the tertiary folding of a protein . Epitopes formed from contiguous amino acids are typically retained by exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost in treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a single spatial conformation.

Modification-assisted profiling (MAP), also known as antibody profiling based on the structure of the antigen (ASAP), is a method that categorizes a large number of monoclonal antibodies (mAb) directed against the same antigen according to the similarities of the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (US 2004/0101920). Each category may reflect a clearly different or partially overlapped single epitope with the epitope represented by another category. This

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technology allows rapid filtering of genetically identical mAbs, so that the characterization can focus on genetically distinct mAbs. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce mAbs that have the desired characteristics. MAP can be used to classify the anti-ANGPTL4 mAbs of the invention into groups of mAbs that bind different epltopos.

ANGPTL4 contains a spiral coil domain at the amino terminal end and a fibrinogen-like domain at the carboxyl terminal end and the full-length ANGPTL4 protein forms an oligomer supported by intermolecular disulfide bonds (Ge et al., 2004, J Bio Chem 279 (3): 2038-2045). It has been reported that the spiral coil domain of the N-terminal end mediates the oligomerization of ANGPTL4 (Ge et al., Cited above) and is also important in the inhibition of LPL activity (Ge et al., 2005, J Lipid Res. 46: 1484-1490; and Ono et al., 2003, J Biol Chem. 278: 41804-41809). The anti-hANGPTL4 antibody or antigen-binding fragment of an antibody binds to an epltope within the spiral coil domain of the N-terminus (residues 1-123) of hANGPTL4 (SEQ ID NO: 476). An anti-hANGPTL4 antibody or fragment thereof can bind an epltope within the region from about residue 1 to about residue 25, from about residue 25 to about residue 50, from about residue 50 to about residue 75, from about residue 75 to about residue 100, from about residue 100 to about residue 125, from about residue 125 to about residue 150, of hANGPTL4 (SEQ ID NO: 476). The antibody or antibody fragment may bind to an epltope that includes more than one of the epltopos listed within the spiral coil domain of the N-terminus of hANGPTL4. The hANGPTL4 antibodies or fragments thereof can bind to one or more fragments of hANGPTL4, for example, a fragment of residues 26 to 406, residues 26 to 148, residues 34 to 66 (antibodies of the invention) and / or residues 165 to 406, of SEQ ID NO: 476.

It can be easily determined whether an antibody binds to the same epltopo or competes for binding with a reference anti-hANGPTL4 antibody using routine methods known in the art. For example, to determine whether a test antibody binds to the same epltopo as a reference anti-hANGPTL4 antibody of the invention, the reference antibody is allowed to bind to a hANGPTL4 peptide or protein under saturation conditions. Next, the ability of a test antibody to bind to the hANGPTL4 molecule is evaluated. If the test antibody is capable of binding to hANGPTL4 after saturation binding with the reference anti-hANGPTL4 antibody, it can be concluded that the test antibody binds to an epltope other than the reference anti-hANGPTL4 antibody. On the other hand, if the test antibody is not able to bind to the hANGPTL4 molecule after saturation binding with the reference anti-hANGPTL4 antibody, then the test antibody can bind to the same epltopo as the antibody bound epltopo reference anti-hANGPTL4 of the invention.

To determine whether an antibody competes for binding with a reference anti-hANGPTL4 antibody, the binding methodology described above is carried out in two orientations: In a first orientation, the reference antibody is allowed to bind to a hANGPTL4 molecule. under saturation conditions followed by the evaluation of the binding of the test antibody to the hANGPTL4 molecule. In a second orientation, the test antibody is allowed to bind a molecule of hANGPTL4 under saturation conditions, followed by evaluation of the binding of the reference antibody to the ANGPTL4 molecule. If, in both orientations, only the first (saturation) antibody is capable of binding to the ANGPTL4 molecule, then it is concluded that the test antibody and the reference antibody compete for hANGPTL4 binding. As one skilled in the art will appreciate, an antibody that competes for binding with a reference antibody may not necessarily bind to the identical epltope as the reference antibody, but may sterically block the binding of the reference antibody by binding a adjacent superposition or epltopo.

Two antibodies bind to the same epltopo or epltopo of superposition if each competitively inhibits (blocks) the union of the other with the antigen. That is, an excess of 1, 5, 10, 20 or 100 times of one antibody inhibits the binding of the other by at least 50%, but preferably 75%, 90% or even 99%, as measured in a binding assay. competitive (see, for example, Junghans et al., Cancer Res, 1990: 50: 1495-1502). Alternatively, two antibodies have the same epltopo if essentially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other. Two antibodies have overlapping epltopos if some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other.

An additional routine experimentation (eg, peptide mutation and binding analysis) can then be carried out to confirm if the observed lack of binding of the test antibody is actually due to binding to the same epltope as the reference antibody or if the steric block (or other phenomenon) is responsible for the lack of union observed. Experiments of this type can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative antibody binding assay available in the art.

Immunoconjugates

Human anti-ANGPTL4 monoclonal antibodies conjugated to a therapeutic fraction (immunoconjugate), such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant or a radioisotope are contemplated. Cytotoxin agents include any agent that is harmful to cells. Examples of suitable cytotoxic agents and chemotherapeutic agents for forming immunoconjugates are known in the art, see, for example, WO 05/103081.

Bispeclophic

The antibodies of the present invention can be monospecific, bispecific or multispecific. Multispecific mAbs may be specific for different epltopos of a target polypeptide or may contain antigen binding domains, specific for more than one target polypeptide. See, for example, Tutt and 10 collaborators (1991) J. Immunol. 147: 60-69. Human anti-hANGpTL4 mAbs may be linked or coexpressed with another functional molecule, for example, another peptide or protein. For example, an antibody or fragment thereof may be functionally linked (for example, by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody with a second binding specificity.

An example of a bispecific antibody format that can be used in the context of the present invention involves the use of a first immunoglobulin (Ig) Ch3 domain and a second Ig CH3 domain, in which the first and second Ig Ch3 domains differ each other in at least one amino acid, and in which at least one amino acid difference reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody 20 that lacks the amino acid difference. In one embodiment, the first Ch3 domain of Ig binds to protein A and the second Ch3 domain of Ig contains a mutation that reduces or suppresses binding to protein A such as a modification of H95R (by exon numbering IMGT; H435R by EU numbering). The second Ch3 may also comprise a modification of Y96F (by IMGT; Y436F by EU). Other modifications that can be found in the second Ch3 include: D16E, L18M, N44S, K52N, V57M and V82I (by IMGT; D356e, 25 L358M, N384S, K392N, V397M and V422I by EU) in the case of IgG1 antibodies; N44S, K52N and V82I (IMGT; N384S,

K392N, and V422I by EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I by EU) in the case of IgG4 antibodies. Variations in the bispecific antibody format described above are contemplated.

Bioequivalent

The anti-hANGPTL4 antibodies and antibody fragments of the present invention encompass proteins that have amino acid sequences that vary from those of the described mAbs, but retain the ability to bind human ANGPTL4. Such mAb variants and antibody fragments comprise one or more amino acid additions, deletions or substitutions when compared to the original sequence, but exhibit biological activity that is essentially equivalent to that of the described mAbs. Similarly, the DNA sequences encoding the hANGPTL4 antibody of the present invention encompass sequences comprising one or more nucleotide additions, deletions or substitutions when compared to the described sequence, but encoding an anti-antibody antibody fragment. -hANGPTL4 which is essentially bioequivalent to an anti-hANGPTL4 antibody or antibody fragment of the invention. Examples of such amino acid sequences and DNA variants were discussed above.

40 Two proteins or antibodies that bind antigens are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose speed and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single dose or multiple doses. Some antibodies will be considered equivalent or pharmaceutical alternatives if they are equivalent in the degree of their absorption, but not in their absorption rate and, nevertheless, they can be considered bioequivalent because these differences in the absorption rate are intentional and are reflected in the labeling. , are not essential to achieve effective concentrations of drugs in the body, for example, in chronic use, and are considered medically insignificant for the particular drug studied.

In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity and potency.

50 Two antigen-binding proteins can be bioequivalent if a patient can be changed one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity or decrease. of efficacy, compared with continued therapy without such change.

Two antigen-binding proteins can be bioequivalent if both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.

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Bioequivalence can be demonstrated by in vivo and in vitro methods. Bioequivalence measures include, for example, (a) an in vivo test in humans or other mammals, in which the concentration of the antibody or its metabolites is measured in blood, plasma, serum or other biological fluid as a function of time; (b) an in vitro test that has been correlated and is reasonably predictive of human bioavailability data in vivo; (c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its objective) is measured as a function of time; and (d) in a well-controlled clinical trial that establishes the safety, efficacy, or bioavailability or bioequivalence of an antibody.

The bioequivalent variants of anti-hANGPTL4 antibodies of the invention can be constructed, for example, by performing various substitutions of residues or sequences or by eliminating terminal or internal residues or sequences not necessary for biological activity. For example, cystel residues that are not essential for biological activity can be removed or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges after renaturation.

Therapeutic administration and formulations

The invention provides therapeutic compositions comprising anti-hANGPTL4 antibodies or antigen binding fragments thereof of the present invention and antibodies for use in therapeutic methods. The administration of therapeutic compositions according to the invention will be carried out with suitable vehicles, excipients and other agents that are incorporated into the formulations to provide a better transfer, delivery, tolerance and the like. A multitude of appropriate formulations can be found in the prescription known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicles containing lipids (cationic or anionic) (such as LIPOFECTINMR), DNA conjugates, anhydrous absorption pastes, oil-in-water emulsions. and water in oil containing Carbowax (polyethylene glycols of various molecular weights), semi-solid gels and semi-solid mixtures containing Carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52: 238-311.

The dose may vary depending on the age and size of the subject to be administered, the target disease, the purpose of the treatment, the conditions, the route of administration and the like. When the antibody of the present invention is used to treat various conditions and diseases directly or indirectly associated with ANGPTL4, including hypercholesterolemia, disorders associated with LDL and apolipoprotelnum B, and lipid metabolism disorders, and the like, in an adult patient, it is advantageous to administer intravenously or subcutaneously the antibody of the present invention in a single dose of about 0.01 to about 20 mg / kg body weight, more preferably from about 0.02 to about 7, about 0.03 to about 5 or about 0.05 to about 3 mg / kg body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. The antibody or antigen binding fragment thereof of the invention can be administered as an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 500 mg, about 5 to about 300 mg, or about 10 up to about 200 mg, up to about 100 mg, or up to about 50 mg. The initial dose may be followed by the administration of a second or several subsequent doses of the antibody or antigen binding fragment thereof in an amount that may be approximately equal to or less than that of the initial dose, where subsequent doses are separated. for at least 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

Several delivery systems are known and can be used to administer the pharmaceutical composition of the invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al. (1987) J. Biol. Chem. 262: 4429-4432). The introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral vlas. The composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous coatings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with other agents. biologically active The administration can be systematic or local.

The pharmaceutical composition can also be supplied in a vesicle, in particular a liposome (see Langer (1990) Science 249: 1527-1533, Treat et al. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler ( eds.), Liss, New York, pages 353-365, Lopez-Berestein, in the same place, pages 317-327, see generally in the same place).

In certain situations, the pharmaceutical composition can be administered in a controlled release system. A pump can be used (see Langer, cited above, Sefton (1987) CRC Crit. Ref. Biomed Eng. 14: 201). Polymeric materials can also be used; see, Medical Applications of Controlled Release, Langer and Wise

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(eds.), CRC Pres., Boca Raton, Florida (1974). A controlled release system can be placed in the vicinity of the objective of the composition, thus requiring only a fraction of the systematic dose (see, for example, Goodson, in Medical Applications of Controlled Release, cited above, volume 2, pages 115138, 1984).

Injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations can be prepared by publicly known methods. For example, injectable preparations can be prepared, for example, by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As an aqueous medium for injections, there is, for example, a physiological saline solution, an isotonic solution containing glucose and other auxiliary agents, etc., which can be used in combination with an appropriate solubilizing agent such as an alcohol (for example, ethanol ), a polyalcohol (for example, propylene glycol, polyethylene glycol), a non-ionic surfactant [eg, polysorbate 80, HCO-50 (polyoxyethylene adduct (50 moles) of hydrogenated castor oil)], etc. As an oily medium, for example, sesame oil, soybean oil, etc., which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. are used. The injection thus prepared is preferably filled in an appropriate vial. A pharmaceutical composition of the present invention can be administered subcutaneously or intravenously with a needle and a standard syringe. In addition, with respect to subcutaneous administration, a pen-type delivery device easily has applications in the administration of a pharmaceutical composition of the present invention. Such pen supply device can be reusable or disposable. A reusable pen type administration device generally uses a replaceable cartridge containing a pharmaceutical composition. Once the entire pharmaceutical composition has been administered inside the cartridge and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen type supply device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Instead, the disposable pen delivery device is preloaded with the pharmaceutical composition retained in a reservoir within the device. Once the deposit is emptied of the pharmaceutical composition, the entire device is discarded.

Numerous reusable pen and auto-injector supply devices have applications in the subcutaneous administration of a pharmaceutical composition of the present invention. Examples include, but are certainly not limited to, AUTOPENMR (Owen Mumford, Inc., Woodstock, RU), DIsEtRONICmr pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75 / 25MR pen, HUMALOGMR pen, HUMALIN 70 / 30MR pen (Eli Lilly and Co., Indianapolis, IN), NOVOPENMR I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIoRmr (Novo Nordisk, Copenhagen, Denmark), BDmr pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPENmr, OPTIPEN PROMR, OPTIPEN STARLETMR and OPTICLIKMR (Sanofi-Aventis, Frankfurt, Germany), to name just a few. Examples of disposable pen delivery devices that have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention include, but are certainly not limited to, the SOLOSTARMR (Sanofi-Aventis), FLEXPENMR (Novo Nordisk) and KWIKPENMR (Eli) pen. Lilly)

Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in dosage forms in a unit dose suitable for adjusting a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforementioned antibody generally contains about 0.1 to about 800 mg per dosage form in a unit dose; especially as an injection, the aforementioned antibody is contained in about 1 to about 500 mg, in about 5 to 300 mg, in about 8 to 200 mg and in about 10 to about 100 mg for the other dosage forms.

Combined therapies

The invention further provides antibodies for use in therapeutic methods for the treatment of diseases or disorders, which are directly or indirectly associated with hANGPTL4, by administering the hANGPTL4 antibody or fragment thereof of the invention in combination with one or more additional therapeutic agents. The additional therapeutic agent may be one or more of any agent that is advantageously combined with the antibody or fragment thereof of the invention, including HMG-CoA reductase inhibitors, such as cerovastatin, atorvastatin, simvastatin, pitavastine, rosuvastatin, fluvastatin, lovastatin. , pravastatin and the like; niacin; various fibrates, such as fenofibrate, bezafibrate, ciprofibrate, clofibrate, gemfibrozil and the like; LXR transcription factor activators, and the like. In addition, the hANGPTL4 antibody or fragment thereof of the invention can be co-administered with other ANGPTL4 inhibitors, as well as with other molecule inhibitors, such as ANGPTL3, ANGPTL5, ANGPTL6 and proprotein convertase subtilisin / kexin type 9 (PCSK9), which they are involved in lipid metabolism, in particular, cholesterol and / or triglyceride homeostasis. Inhibitors of these molecules include small molecules and antibodies that specifically bind to these molecules and block their activity (see, for example, the anti-PCSK9 antibodies described in US 2010/0166768 A1).

In addition, the additional therapeutic agent may be one or more anticancer agents, such as chemotherapeutic agents, antiangiogenic agents, growth inhibiting agents, cytotoxic agents, apoptotic agents and other agents well known in the art for treating cancer or other proliferative diseases or disorders. . Examples of anticancer agents include, but are not limited to, an antifungal agent, such as docetaxel, paclitaxel and the like; a platinum-based chemotherapeutic compound, such as cisplatin,

carboplatin, iproplatin, oxaliplatin and the like; or other conventional cytotoxic agents such as 5-fluorouracil, capecitabine, irinotecan, leucovorin, gemcitabine and the like, and antiangiogenic agents, including vascular endothelial growth factor (VEGF) antagonists, such as anti-VEGF antibodies, for example bevacizumab (AVASTIN®) , Genentech) and a VEGF receptor-based blocker, for example, "10 VEGF trap" described in US Patent No. 7,070,959, delta type 4 ligand antagonists (DII4), such as anti-antibody -DII4 as described in the publication of US patent application No. 2008/0181899, and a fusion protein containing the extracellular domain of DII4, for example, DII4-Fc as described in the publication of the patent application United States No. 2008/0107648; receptor tyrosine kinase inhibitors and / or angiogenesis, including sorafenib (NEXAVAR® from Bayer Pharmaceuticals Corp.), sunitinib 15 (SUTENT® from Pfizer), pazopanib (VOTRIENTMR from GlaxoSmithKline), toceranib (PALLADIAMR from Pfizer),

vandetanib from Astra-Zeneca), cediranib (RECENTIN® from Astra-Zeneca), regorafenib (BAY 73-4506 from Bayer), axitinib (AG013736 from Pfizer), lestaurtinib (CEP-701 from Cephalon), erlotinib (TARCEVA® from Genentech) , gefitinib (Astra-Zeneca IRESSAMR), BIBW 2992 (TOVOK® from Boehringer Ingelheim), lapatinib (TYKERB® from GlaxoSmithKline), neratinib (HKI-272 from Wyeth / Pfizer), and the like, and salts, acids or pharmaceutically derived 20 acceptable from any of the above. In addition, other therapeutic agents, such as analgesics, anti-inflammatory agents, including non-steroidal anti-inflammatory drugs (NSAIDs), such as Cox-2 inhibitors, and the like, can also be co-administered with the hANGPTL4 antibody or fragment thereof of the invention with in order to improve and / or reduce the symptoms that accompany the underlying cancer / tumor.

The hANGPTL4 antibody or fragment thereof of the invention and the additional therapeutic agent or agents may be administered together or separately. When separate dosage formulations are used, the antibody or fragment thereof of the invention and the additional agents may be administered concurrently or separately at staggered intervals, that is, sequentially, in appropriate orders.

Diagnostic uses of antibodies

The anti-ANGPTL4 antibodies of the present invention can also be used to detect and / or measure ANGPTL4 30 in a sample, for example, for diagnostic purposes. For example, an anti-ANGPTL4 Ab or fragment thereof can be used to diagnose a condition or disease characterized by aberrant expression (eg, overexpression, subexpression, lack of expression, etc.) of ANGPTL4. Examples of diagnostic tests for ANGPTL4 may comprise, for example, contacting a sample obtained from a patient, with an anti-ANGPTL4 antibody of the invention, wherein the anti-ANGPTL4 antibody is labeled with a detectable marker or a Reporter molecule or is used to selectively capture and isolate ANGPTL4 from patient samples. Alternatively, an unlabeled anti-ANGPTL4 Ab can be used in diagnostic applications in combination with a secondary antibody that is detectably labeled. The detectable marker or reporter molecule may be a radioisotope, such as 3H, 14C, 32P, 35S, 131I or 125I; a fluorescent or chemiluminescent fraction, such as fluorescell isothiocyanate, or rhodamine; or an enzyme such as alkaline phosphatase, p-galactosidase, horseradish peroxidase or luciferase. Assays that can be used to detect or measure ANGPTL4 in a sample include enzymatic immunoabsorption assays (ELISA), radioimmunoassay (RIA), fluorescence activated cell sorting (FACS), and the like.

Examples

Efforts have been made to ensure accuracy with respect to the numbers used, but some experimental errors and deviations should be explained. Unless otherwise indicated, the molecular weight is the average molecular weight, the temperature is in degrees Celsius and the pressure is at or near atmospheric.

Example 1: Generation of human antibodies with human ANGPTL4

VELOCIMMUNEmr mice were immunized with human ANGPTL4, and the antibody immune response was monitored by an antigen specific immunoassay using serum obtained from these mice. 50 B cells expressing anti-hANGPTL4 were collected from the spleens of immunized mice that have been shown to have elevated anti-hANGPTL4 antibody titers and fused with mouse myeloma cells to form hybridomas. Hybridomas were screened and selected to identify cell lines expressing hANGPTL4 specific antibodies using assays as described below. The assays identified several cell lines that produced chimeric anti-HANGPTL4 antibodies designated as H1M222, H1M223, H1M224, H1M225, H1M234 and H1M236.

Human ANGPTL4 specific antibodies were also isolated directly from B cells immunized with antigen without fusion to myeloma cells, as described in US-A-2007/0280945 A1. The

Heavy and light chain variable regions were cloned to generate fully human antihANGPTL4 antibodies designated H1H257, H1H268, H1H283, H1H291, H1H295, H1H291, H1H292, H1H295, H1H624, H1H637, H1H638, H1H644. CHO cell lines expressing recombinant antibody were established.

5 Example 2. Analysis of the use of variable genes

To analyze the structure of the antibodies produced, nucleic acids encoding antibody variable regions were cloned and sequenced. From the nucleic acid sequence and the predicted amino acid sequence of the antibodies, the use of genes for each heavy chain variable region (HCVR) and light chain variable region (LCVR) was identified. Table 1 shows the use of genes for antibodies selected in accordance with the invention.

Table 1

 Antibody

 HCVR LCVR

 Vh

 Dh Jh Vk Jk

 H1M225

 3-9 1-7 1 1-5 2

 H1M236

 3-9 6-6 5 3-15 5

 H1H283

 3-13 1-26 4 1-5 1

 H1H285

 3-13 1-26 4 1-5 1

 H1 H291

 3-13 1-26 4 1-5 1

 H1H292

 3-13 1-26 4 1-5 1

 H1H295

 3-13 1-26 4 1-5 1

 H1H637

 3-13 1-26 4 1-5 1

 H1H638

 3-13 1-26 4 1-5 1

 H1H644

 3-13 1-26 4 1-5 1

 H1H257

 3-13 1-26 4 1-5 1

 H1M224

 3-13 3-3 4 1-16 3

 H1M223

 3-15 3-3 4 1-12 3

 H1M234

 3-15 3-3 4 1-12 3

 H1H624

 3-23 5-5 6 1-9 3

 H1H284

 3-30 5-12 6 1-9 4

 H1M222

 3-33 3-9 5 3-20 4

 H1H653

 3-33 2-8 6 1-17 2

 H1H268

 4-34 3-3 4 1-27 4

Table 2 shows the amino acid sequence pairs of the heavy and light chain variable region of selected anti-hANGPTL4 antibodies and their corresponding antibody identifiers. The

N, P and L designations refer to antibodies that have heavy and light chains with identical CDR sequences, but with sequence variations in regions that fall outside the CDR sequences (i.e., in structural regions). Therefore, the N, P and L variants of a particular antibody have identical CDR sequences within their heavy and light chain variable regions, but contain modifications within the 5 structural regions.

Table 2

 Name

 HCVR / LCVR SEQ ID NOs Name HCVR / LCVR SEQ ID NOs Name HCVR / LCVR SEQ ID NOs

 H1M222N

 314/322 H1M222P 330/332 H1M222L 334/336

 H1M223N

 410/418 H1M223P 426/428 H1M223L 430/432

 H1M224N

 338/346 H1M224P 354/356 H1M224L 358/360

 H1M225N

 362/370 H1M225P 378/380 H1M225L 382/384

 H1M234N

 434/442 H1M234P 450/452 H1M234L 454/456

 H1M236N

 386/394 H1M236P 402/404 H1M236L 406/408

 H1H236N2

 458/394 H1H236P2 466/404 H1H236L2 468/408

 H1H257N

 2/10 H1H257P 18/20 H1H257L 22/24

 H1H268N

 26/34 H1H268P 42/44 H1H268L 46/48

 -

 -

 H4H268P2 487/44

 -

 -

 H1H283N

 50/58 H1H283P 66/68 H1H283L 70/72

 H1H284N

 74/82 H1H284P 90/92 H1H284L 94/96

 H1H285N

 98/106 H1H285P 114/116 H1H285L 118/120

 H1H291N

 122/130 H1H291P 138/140 H1H291L 142/144

 H1H292N

 146/154 H1H292P 162/164 H1H292L 166/168

 H1H295N

 170/178 H1H295P 186/188 H1H295L 190/192

 H1H624N

 194/202 H1H624P 210/212 H1H624L 214/216

 H1H637N

 218/226 H1H637P 234/236 H1H637L 238/240

 H1H638N

 242/250 H1H638P 258/260 H1H638L 262/264

 H1H644N

 266/274 H1H644P 282/284 H1H644L 286/288

 H1H653N

 290/298 H1H653P 306/308 H1H653L 310/312

Example 3: Determination of the binding affinity of hANGPTL4

The equilibrium dissociation constants (Kd values) were determined for the binding of the antigen to selected antibodies that bind amino acid residues 26-148 of human fused line ANGPTL4 with

Mouse IgG2a (hANGPTL4-mFc; SEQ ID NO: 480) by surface kinetics using a real-time biosensor surface plasmon resonance assay (BIACOREMR T100). HANGPTL4-mFc was captured with goat anti-mouse IgG polyclonal antibody (GE Healthcare) that was chemically coupled to a BIACOREMR chip through free amino groups. Variable concentrations (from 12.5 nM to 50 nM) of anti-ANGPTL4 antibodies were injected onto the surface of the captured antigen for 90 seconds. Antigen-antibody binding and dissociation were monitored in real time at 25 ° C and 37 ° C. A kinetic analysis was performed to calculate Kd and the half-life of the antigen / antibody complex dissociation. The results are shown in Table 3. A human anti-EGFR antibody was used as a negative control, which showed no binding to the captured hANGPTL4-mFc.

Table 3

 Antibody

 25 ° C 37 ° C

 Kd (pM)

 T1 / 2 (min) Kd (pM) t1 / 2 (min)

 H1H257P

 201 91 238 63

 H1H268P

 275 80 389 57

 H1H283P

 130 119 1360 12

 H1H284P

 168 162 349 81

 H1H285P

 92.5 156 194 71

 H1H291P

 87.6 303 178 122

 H1H292P

 136 112 167 88

 H1H295P

 30.7 874 2620 10

 H1H624P

 1190 7 3710 3

 H1H638P

 193 85 299 48

 H1H644P

 111 144 3000 6

 H1H653P

 411 43 2130 6

10

For H1 H268P and H1 H284P, Fab fragments were prepared by papal digestion and purified by standard purification methods, and their binding affinities to hANGPTL4 were measured at 25 ° C at pH 7.2 and pH 5.75 using the system BIACOREMR, essentially in accordance with the method described above. Simply put, several concentrations (3,125 nM-100 nM) of anti-hANGPTL4 antibodies (i.e., Fab H1 15 H268, mAb H1 H268, Fab H1 H284 and mAb H1 H284) were injected over a surface of hANGPTL4 (26 -148) -mFc

captured with low density anti-mFc (~ 68 ± 4 RU), or the surface of hANGPTL4 (26-406) -His coupled to amino (R&D Systems) (450 RU) or the N-terminal end region of mono of amino-coupled Cynomolgus (amino acid residues 1-130 of SEQ ID NO: 490) expressed with a hexa-histidine tag at the C-terminal end (MfANGPTL4 (1-130) -His) (1,028 RU). A kinetic analysis was performed to measure the values of ka and kd, and 20 the values of Kd and the half-life of the antigen / antibody complex dissociation were calculated. The results are shown in Table 4 (H1 H268P) and in Table 5 (H1 H284P).

Table 4

 Antigen

 Antibody H1H268P pH Captured antigen (RU) mAb 50 mM or Fab bound ka (M-V) kd (s-1) Kd (pM) t1 / 2 (min)

 hANGPTL4

 mAb integer 7.2 35 ± 1.9 40 1.53 x 9.59 629 120

 (26-148) -mFc

         105 x 10-5

 5.75

 27 ± 0.6 77 6.28 x 105 1.38 x 10-4 220 84

 Fab

 7.2 35 ± 1.9 10 3.00 x 105 6.01 x 10-4 2,000 19

 5.75

 27 ± 0.6 20 1.74 x 105 3.04 x 10-3 17.5 (nM) 4

 HANGPTL4 (26-406) -His

 Integer mAb 7.2 450 RU coupled with amino 38 4.89 x 105 2.00 x 10-4 408 58

 5.75

 45 9.23 x 105 4.46 x 10-4 483 26

 Fab

 7.2 13 7.26 x 105 1.18 x 10-2 16.3 (nM) 1

 5.75

 10 4.44 x 105 6.57 x 10-3 14.8 (nM) 2

 MfANGPTL4 (1-130) -His

 Integer mAb 7.2 1.028 RU coupled with amino 279 3.92 x 105 4.76 x 10-5 122 243

 5.75

 583 1.07 x 105 8.24 x 10-5 77.2 140

 Fab

 7.2 167 2.67 x 105 1.71 x 10-3 6.420 7

 5.75

 178 3.12 x 105 4.32 x 10-3 13.8 (nM) 3

Table 5

 Antigen

 Antibody H1H284P PH Captured antigen (RU) mAb 50 mM or Fab bound ka (M-V) kd (s-1) Kd (pM) t1 / 2 (min)

 hANGPTL4 (26-148) -mFc

 mAb integer 7.2 35 ± 1.9 99 2.74 x 105 5.36 x 10-5 196 216

 5.75

 27 ± 0.6 171 1.09 x 106 8.91 x 10-5 81.9 130

 Fab

 7.2 35 ± 1.9 29 2.45 x 105 2.02 x 10'4 823 57

 5.75

 27 ± 0.6 56 4.72 x 105 1.60 x 10-3 3,400 7

 hANGPTL4

 mAb integer 7.2 450 RU coupled 77 8.50 x 8.85 105 130

 (26-406) -His

     with amino 105 x 10-5

 5.75

 101 1.93 x 106 2.72 x 10-4 141 42

 Fab

 7.2 32 1.11 x 106 3.44 x 10-4 310 34

 5.75

 33 1.21 x 106 1.73 x 10-3 1.440 7

 MfANGPTL4 (1-130) -His

 Integer mAb 7.2 1.028 RU coupled with amino 414 4.67 x 105 5.83 x 10-5 125 198

 5.75

 804 1.55 x 106 8.42 x 10-5 54.3 137

 Fab

 7.2 214 3.10 x 105 3.13 x 10-3 10.1 (nM) 4

 5.75

 255 7.24 x 105 6.19 x 10-3 8.540 2

Both Fab fragments were capable of binding to all forms of ANGPTL4, although with lower affinities than complete antibody molecules.

Example 4. Determination of cross-reactivity of anti-hANGPTL4 antibodies

5 The possible cross-reactivity of anti-hANGPTL4 antibodies with related proteins, ie hANGPTL3, human angiopoietin-like protein (hANGPTL5) and mouse ANGPTL4 (mANGPTL4) for the selected antibodies, ie H1 H268P and H1 H284P, was tested. , using the BIACOREMR system. Simply put, anti-hANGPTL4 as! Antibodies were injected. as negative controls, that is, two monoclonal antibodies (Control a and Control b) that are not binders with any ANGPTL protein, at a rate of 3,125 pg / mL-50 pg / mL 10 on chip surfaces coupled with hANGPTL3-His amine ( R&D Systems, cat. # 3829-AN) to 5228 RU, hANGPTL4-His (R & D Systems, cat. # 4487-AN) to 6247 RU, hANGPTL5-His (Abnova Corp., cat. # H00253935-P01 ) at 5265 RU, and mANGPTL4-His [R26-S410 of mANGPTL4 (SEQ ID NO: 478) fused with an AS linker to a 6-histidine tag at the C-terminus] at 5233 RU, respectively. A polyclonal antibody specific for hANGPTL3 was also tested (R&D Systems, cat. # BAF3485). The binding of each antibody, expressed as a specific RU value, was determined, and the results are shown in Table 6.

Table 6

 injected mAb

 Specific RU

 hANGPTL3-His

 hANGPTL4-His hANGPTL5-His mANGPTL4-His

 Regulator

 -17 -23 -18 -7

 H1H268P

 -17 768 -16 -7

 H1H284P

 -6 1351 -13 18

 Control a

 -16 -23 -18 -6

 Control b

 -17 -23 -18 -6

5

10

fifteen

twenty

25

30

35

40

 Anti-hANGPTL3

 680 -1 -1 2319

H1 H268P and H1 H284P only specifically joined hANGPTL4-His and did not join any of the other related ANGPTL proteins.

In addition, the binding affinities of H1 H268P and H1 H284P for various ANGPTL3 and ANGPTL4 peptides were also determined by the BIACOREMR system. In summary, H1 H268P (1348 ± 11 RU) and H1 H284P (868 ± 13 RU) were captured on an anti-human Fc surface and various concentrations (62.5 nM-500 nM) of peptides hANGPTL3 and hANGPTL4 were injected . The peptides tested were hANGPTL4 (R34-L66 of SEQ ID NO: 476), biotinylated hANGPTL4 at the N-terminal end (R34-L66 of SEQ ID NO: 476), hANGPTL3 (R36-I68 of SeQ ID NO: 485) , and biotinylated HANGPTL3 at the N-terminal end (R36-I68 of SeQ ID NO: 485). A kinetic analysis was performed to measure ka and kd, and the values of Kd and the half-life of the antigen / antibody complex dissociation were calculated. The results are shown in Table 7. NB: There is no union under the experimental conditions described.

Table 7

 Anti-hANGPTL4 antibody

 Peptide ka (M'V) kd (s-1) Kd (pM) t1 / 2 (min)

 hANGPTL3-Nterm biotin NB - - -

 H1H268P

 hANGPTL4-Nterm biotin 4.53 x 103 2.94 x 10-4 64.8 39

 hANGPTL3 NB - - -

 hANGPTL4 6.49 x 103 3.65 x 10-4 56.3 32

None of the antibodies bound to any of the hANGPTL3 peptides. In addition, H1 H284P did not bind to any of the hANGPTL4 peptides even at the highest peptide concentration tested (500 nM), while H1 H268P was able to bind both hANGPTL4 peptides. This suggests that H1 H268P recognizes a linear epltopo within region 34-66. In contrast, H1 H284P either joins outside this region or does not recognize a linear epltopo in this region.

Example 5. Inhibition of hANGPTL4 by anti-ANGPTL4 antibodies

Lipoprotelna lipase (LPL) plays a critical role in the metabolism of lipids in humans. LPL catalyzes the hydrolysis of triglycerides and releases fatty acids to be metabolized. ANGPTL4 inhibits the activity of LPL that leads to an increase in the level of lipids (Oike et al., 2005, Trends in Molecular Medicine 11 (10): 473-479). The spiral coil region of the N-terminal end of ANGPTL4 undergoes homo-multimerization, both in isolation and when they bind to the fibrinogen region of the C-terminus. The N-terminal region also inhibits LPL when expressed without the fibrinogen region of the C-terminus. A bioassay without cells was developed to determine the ability of selected anti-hANGPTL4 antibodies to inhibit the decrease induced by ANGPTL4 in LPL activity.

Inhibition of hANGPTL4 activity by selected anti-hANGPTL4 antibodies was determined using the CONFLUOLIPMR continuous fluorometric lipase test (Progen, Germany) using two hANGPTL4 proteins: full length hANGPTL4 (i.e. amino acid residues 26-406 of the SEQ ID NO: 476) with a hexa-histidine tag at the C-terminal end (hANGPTL4-His; R & D Systems, MN) and hANGPTL4-mFc (SEQ ID NO: 480) that contains the spiral coil region of the terminal end N.

Briefly, 2 pM bovine LPL, 0.25 pM human ApoClI (a cofactor of LPL), 2 mg / mL of BSA and 1.6 mM CaCh, were previously mixed in a 96-well test plate. Either the hANGPTL4-His or hANGPTL4-mFc protein was added to the Apo / LPL mixture to a final concentration of 10 nM and 2 nM, respectively. Next, mixtures of Apo / LPL / ANGPTL4 proteins were added together with anti-hANGPTL4 antibodies diluted in series with an initial concentration of 300 nM (for the inhibition of hANGPTL4-His) or 100 nM (for the inhibition of hANGPTL4-mFc ) and incubated at room temperature for 30 minutes (final volume of 50 pl). After incubation, 200 µl of reconstituted lipase substrate, 1-trinitrophenyl-amino-dodecanoyl-2- pirendecanoyl-3-0-hexadecyl-sn-glycerol (LS-A, Progen) was added to the antibody mixture and incubation at 37 ° C for two hours. The fluorescence was then measured at 342 nm / 400 nm (excitation / emission) using a microplate reader

FlexStation® 3 (Molecular Devices, CA). The fluorescence is directly proportional to the activity of LPL. The results are shown in Table 8. Control I: a rabbit polyclonal antibody specific for hANGPTL4 (BioVendor). Control II: an irrelevant human antibody that does not bind to hANGPTL4. NT: not tested. Total inhibition (i.e. 100% inhibition) was determined from the relative fluorescence unit (RFU) of assay 5 with 2 nM bovine LPL, 0.25 pM human ApoCll, 2 mg / ml BSA and 1.6 mM CaCl2, ANGPTL4 and ANGPTL4 proteins.

Table 8

 Antibody

 % inhibition of hANGPTL4 activity

 hANGPTL4-mFc (2 nM)

 hANGPTL4-His (10 nM)

 H1H236N2

 13 19

 H1H257P

 16 NT

 H1H268P

 76 80

 H1H284P

 82 90

 H1H285P

 28 47

 H1H292P

 20 53

 H1H624P

 62 48

 H1H653P

 55 48

 I control

 29 66

 Control II

 Without inhibition Without inhibition

The H1 H284P and H1 H268P antibodies exhibited the highest inhibition of the inhibitory activity of ANGPTL4 against LPL among the tested antibodies, including the hANGPTL4 polyclonal antibody control. For the H1 H284P and H1 H268P antibodies, the antibody concentrations required for 50% maximum inhibition (IC50) of 2 nM hANGPTL4-mFc were determined to be 0.8 nM and 1.2 nM, respectively. In addition, it was determined that the antibody concentrations required for the maximum 50% inhibition of 10 nM hANGPTL4-His were 0.5 nM and 0.2 nM, respectively.

Similarly, H1 H284P and H1 H268P were tested in the LPL bioassay for their ability to inhibit cross-species orthologs: the N-terminal region of cynomolgus monkey (amino acid residues 26148) expressed with a hexa-histidine tag at the N-terminal end (His-MfANGPTL4; SEQ ID NO: 488) and the full-length mouse ortholog (amino acid residues 26-410 of SEQ ID NO: 478) with a hexa-histidine tag at the C-terminal end (mANGPTL4-His). A full dose response was first performed using the ANGPTL4 protein in the LPL assay to determine the EC50 of ANGPTL4 for each experiment and IC50 determinations for each antibody were then made using constant concentrations of ANGPTL4 protein, as shown in the Table 9. Antibody concentrations varied from 0 to 100 nM. Note: Without blocking.

Claims (13)

5 10 fifteen twenty 25 30 35 40 Four. Five 1. An isolated human antibody or antigen-binding fragment thereof that specifically binds to human angiopoietin-like protein 4, comprising: (a) a region 1 determining heavy chain complexity / region 2 determining heavy chain complementarity / region 3 determining heavy chain complementarity having the amino acid sequence combination of SEQ ID NO: 28/30/32; Y (b) a light chain complementarity determining region 1 / light chain complementarity determining region 2 / light chain complementarity determining region 3 having the amino acid sequence combination of SEQ ID NO: 36/38/40. 2. The isolated human antibody or antigen binding fragment of an antibody of claim 1, comprising a heavy chain variable region selected from SEQ ID NO: 487, 26, 42 and 46. 3. The isolated human antibody or antigen binding fragment of an antibody according to any one of claims 1 or 2, comprising a light chain variable region selected from SEQ iD NO: 44, 34 and 48. 4. The antibody or antigen binding fragment according to any one of claims 1 to 3, comprising a pair of sequences of the heavy chain variable region / light chain variable region selected from SEQ ID NO: 487 / 44, 26/34, 42/44 and 46/48. 5. An isolated human antibody or antigen-binding fragment thereof that specifically binds to human angiopoietin-like protein 4, comprising the sequences of heavy and light chain complementarity determining regions contained in the region sequence pair heavy chain variable / light chain variable region of the antibody or antigen binding fragment according to claim 4. 6. The antibody or antigen-binding fragment according to claim 4, wherein the antibody or antigen-binding fragment reacts cross-linked with prinena 4 similar to cynomolgus mono angiopoietin and with protelna 4 similar to angiopoietin de Rhesus monkey 7. An isolated nucleic acid molecule encoding the antibody or antigen binding fragment according to any of the preceding claims. 8. An expression vector comprising the nucleic acid molecule of claim 7, or a host cell comprising said expression vector. 9. A method of producing a protelna 4 antibody similar to human anti-angiopoietin or antigen-binding fragment thereof, comprising growing the host cell of claim 8 under conditions that allow the production of the antibody or fragment thereof. , and recover the antibody or fragment thereof! produced. 10. A pharmaceutical composition comprising the antibody or antigen binding fragment according to any one of claims 1 to 6, and a pharmaceutically acceptable carrier. 11. A pharmaceutical composition of claim 10, further comprising: (i) one or more additional therapeutic agents selected from an HMG-CoA reductase inhibitor; and cholesterol absorption inhibitor or bile acid reabsorption, or both; an agent that increases the catabolism of lipoprotelnas; an agent that reduces the number of nonfatal cardiac attacks; and an activator of the transcription factor LXR; or (ii) one or more additional therapeutic agents selected from statin, niacin, fibrate and anti-PCSK9 antibody. 12. An antibody or antigen-binding fragment according to any one of claims 1 to 6 for use in a method for improving, improving, inhibiting or preventing a disease or disorder mediated by prothena 4 similar to angiopoietin, wherein The angiopoietin-like protelna-4-mediated disease or disorder is selected from hypertriglyceridemia, hypercholesterolemia, chylomicronemia, atherogenic dyslipidemia, cardiovascular disease or disorder, acute pancreatitis, non-alcoholic steatohepatitis, diabetes and obesity. 13. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 in the manufacture of a medicament for use to improve, relieve, inhibit or prevent a similar disease or disorder mediated by prothena 4. to angiopoietin, where the disease or disorder mediated by prothena 4 similar to angiopoietin is selected from hypertriglyceridemia, hypercholesterolemia, 5 chylomicronemia, atherogenic dyslipidemia, cardiovascular disease or disorder, acute pancreatitis, non-alcoholic steatohepatitis, diabetes and obesity. HCDR H1H268P HCVR {SEQ ID N0! 42} H1H284P HCVR {SEQ ID NO! 90} H1H2.91PHCVR (SEQ ID NO !! 3 8} H1H292P HCVR (SEQ ID NO!! 62} H1H268P HCVR {SEQ ID N0I42} H1H284P HCVR {SEQ ID NOI90} H1H291P HCVR {SEQ ID NO! 138} H1H292P HCVR {SEQ ID NON 62} HIH268P HCVR {SEQ ID N0'42} H1H284P HCVR {SEQ ID NO! 90} H1H291P HCVR {SEQ ID NO !! 38} HI H292P HCVR {SEQ ID NOO 62}

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